Human facilitating cells

ABSTRACT

The present disclosure relates to human facilitating cells (hFC), and methods of isolating, characterizing, and using such hFCs.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation-In-Part application of, and claimsbenefit under 35 U.S.C. §120 to, PCT/US09/003,340 filed Jun. 1, 2009,which claims benefit under 35 U.S.C. §119(e) to U.S. Application No.61/057,724 filed May 30, 2008. This application also claims benefitunder 35 U.S.C. §119(e) to U.S. Application No. 61/374,460 filed Aug.17, 2010.

TECHNICAL FIELD

This invention relates to human facilitating cells, and the use of suchcells in therapeutic protocols.

BACKGROUND

Facilitating cells (FCs) from mouse have been described. See, forexample, U.S. Pat. No. 5,772,994. FCs are not stem cells, butsignificantly improve the initial and long-term engraftment of stemcells. For example, transplantation of stem cells alone only prolongssurvival of a transplant patient, while the presence of FCs result insustained and long-term HSC engraftment. See, also, Kaufman et al.,2005, J. Exp. Med., 201:373-83).

SUMMARY

The present invention relates to human facilitating cells (hFCs),methods of isolating hFCs, and methods of using hFCs for facilitatingreconstitution of a damaged or destroyed hematopoietic system with stemcells as well as for inducing donor-specific tolerance for thetransplantation of donor cells, tissues and solid organs.

In one aspect, a cellular composition comprising is provided. Such acellular composition can include at least about 30% (e.g., at leastabout 40%, at least about 50%, or at least about 60%) human facilitatingcells (hFCs), wherein the hFCs comprise cells having a phenotype ofCD8+/alpha beta TCR−/CD56^(dim/neg) and cells having a phenotype ofCD8+/alpha beta TCR−/CD56^(bright).

In some embodiments, the cells have a phenotype of CD8+/alpha betaTCR−/CD56^(dim/neg) are predominantly CD3 epsilon+/CD19−. In someembodiments, the cells have a phenotype of CD8+/alpha betaTCR−/CD56^(bright) are predominantly CD3 epsilon−/CD19+. In someembodiments, the hFCs include cells having a phenotype of CD8+/alphabeta TCR−/delta gamma TCR+/CD3 epsilon+/CD19+. In some embodiments, thehFCs include cells having a phenotype of CD8+/alpha beta TCR−/B220+/CD11c+/CD11b−. In some embodiments, about 48% of the hFCs are CD8+/alphabeta TCR−/CD3 epsilon+, about 33% of the hFCs are CD8+/alpha betaTCR−/CD19+, about 44% of the hFCs are CD11c+, about 40% of the hFCs areCD11b+, about 42% of the hFCs are Foxp3, and about 30% of the hFCs areHLA-DR. In some embodiments, about 25% of the hFCs are CD8+/alpha betaTCR−/IFN-gamma and about 31% of the hFCs are CD8+/alpha beta TCR−/CXCR4.

In one aspect, a therapeutic cellular composition is provided. Such atherapeutic cellular composition can include human hematopoietic stemcells (HSCs), wherein the HSCs have a phenotype of CD34+; humanfacilitating cells (hFCs), wherein the hFCs comprise cells having aphenotype of CD8+/alpha beta TCR−/CD56^(dim/neg) and cells having aphenotype of CD8+/alpha beta TCR−/CD56^(bright); and human alpha betaTCR+ T cells, wherein the alpha beta TCR+ T cells are present in anamount that is greater than would be considered therapeutic. In someembodiments, the therapeutic cellular composition is for delivery to arecipient. Also in some embodiments, the alpha beta TCR+ T cells arepresent in an amount between about 2.0×10⁶ and about 5.0×10⁶ alpha betaTCR+ T cells/kg recipient body weight. In some embodiments, the numberof alpha beta TCR+ T cells are adjusted to between about 2.0×10⁶ andabout 5.0×10⁶ alpha beta TCR+ T cells/kg recipient body weight. In someembodiments, the number of alpha beta TCR+ T cells are adjusted tobetween about 3.0×10⁶ and about 4.2×10⁶ alpha beta TCR+ T cells/kgrecipient body weight.

In one aspect, a method of making a therapeutic cellular composition fordelivery to a recipient is provided. Such a method typically includesproviding a donor source of hematopoietic stem cells (HSCs); depletingalpha beta TCR+ T cells from the donor source to produce a depleteddonor source; adjusting the number of alpha beta TCR+ T cells in thedepleted donor source to greater than 1×10⁵ alpha beta TCR+ T cells perkg recipient body weight, thereby producing a therapeutic cellularcomposition for delivery to a recipient. In some embodiments, the sourceof HSCs is bone marrow, thymus, peripheral blood, fetal liver, orembryonic yolk sac. In certain embodiments, the T cells are depletedusing one or more antibodies. In some embodiments, the one or moreantibodies are conjugated to magnetic beads. It is a feature of thedisclosure that the hFCs described herein improve the engraftmentability of the HSCs compared to HSCs engrafted in the absence of thehFCs

In one aspect, a method of making the immune system of a recipientchimeric with the immune system of a donor is provided. Such a methodtypically includes administering the therapeutic cellular compositiondescribed above to the recipient, wherein the recipient has beenconditioned.

In some embodiments, the conditioning of the recipient includes a doseof total body irradiation (TBI), wherein the total body irradiation doesnot exceed 300 cGy. In some embodiments, the therapeutic cellularcomposition is administered to the recipient intravenously. In someembodiments, the recipient's immune system is considered to be chimericwith the donor's immune system when the recipient's immune system is atleast about 1% donor origin.

In some embodiments, the recipient has a disease. For example, thedisease can be an autoimmune disease, leukemia, a hemoglobinopathy, aninherited metabolic disorder, or a disease that necessitates an organtransplant. Representative autoimmune diseases include diabetes,multiple sclerosis, and systemic lupus erythematosus. In someembodiments, the disease is an infection by an immunodeficiency virus orhepatitis. In some embodiments, the disease can be a hematopoieticmalignancy, anemia, hemoglobinopathies, or an enzyme deficiency. In someembodiments, the transplanted organ is heart, skin, liver, lung, heartand lung, kidney, pancreas, or an endocrine organ (e.g., a thyroidgland, parathyroid gland, a thymus, adrenal cortex, or adrenal medulla).

In one aspect, a cellular composition is provided that includes at leastabout 30% human facilitating cells (hFCs) having a phenotype ofCD8+/TCR−/CD56^(dim/neg). Such a cellular composition further caninclude hematopoietic stem cells (HSCs), wherein the HSCs have aphenotype of CD34+, wherein the HSCs are MHC-matched with the hFCs. Inanother aspect, a cellular composition is provided that includes humanhematopoietic stem cells (HSCs), wherein the HSCs have a phenotype ofCD34+; and human facilitating cells (hFCs), wherein the hFCs have aphenotype of CD8+/TCR−/CD56^(dim/neg). In one embodiment, the hFCsCD56^(dim/neg) phenotype is CD56^(dim).

According to this disclosure, hFCs further can have a phenotype ofCD3+/CD 16+/CD 19+/CD52+. In addition, hFCs can have a phenotype,without limitation, of CXCR4, CD123, HLADR, NKp30, NKp44, NKp46, CD11c,and CD 162, and hFCs further can be characterized by the presence ofmarkers such as, without limitation, CD11a, CD11b, CD62L, and FoxP3.Typically, the hFCs described herein improve the engraftment ability ofthe HSCs compared to HSCs engrafted in the absence of the hFCs. Cellularcompositions as described herein can include at least about 50% (e.g.,75%, or 90%) of the hFCs. Typically, the phenotype of the cells isdetermined by antibody staining or flow cytometry.

In one aspect, a pharmaceutical composition is provided that includes acellular composition as described herein. In another aspect, methods oftreating a human suffering from a disease are provided. Such methodsgenerally include administering a pharmaceutical composition thatincludes a cellular composition as described herein to a human. In stillanother aspect, methods of transplanting donor cells, tissues, or organsinto a human recipient are provided. Such methods generally includeadministering a pharmaceutical composition that includes a cellularcomposition as described herein to the human recipient.

Such methods can further include partially conditioning the human byexposure to total body irradiation, an immunosuppressive agent, acytoreduction agent, or combinations thereof prior to the administrationof the pharmaceutical composition. In one embodiment, the total bodyirradiation is 200 cGy. A pharmaceutical composition as described hereincan be administered intravenously.

In one embodiment, the disease is an autoimmune disease such asdiabetes, multiple sclerosis, or systemic lupus erythematosus. Inanother embodiment, the disease is an infection by an immunodeficiencyvirus or hepatitis. In yet another embodiment, the disease is chosenfrom a hematopoietic malignancy, anemia, hemoglobinopathies, and anenzyme deficiency.

Representative donor tissues or organs include, without limitation,heart, skin, liver, lung, heart and lung, kidney, pancreas, or anendocrine organ such as a thyroid gland, parathyroid gland, a thymus,adrenal cortex, or adrenal medulla. Donor cells can be pancreatic isletcells, neurons, or myocytes.

In yet another aspect, methods for obtaining a cellular composition thatincludes at least about 0.5% to 8.0% hFCs is provided. Such a methodgenerally includes providing a hematopoietic cell composition; andremoving cells from the hematopoietic cell composition that have aphenotype of alpha beta TCR− and gamma delta TCR−. Such methods canfurther include selecting for cells that have a phenotype of CD8+;selecting for cells that have a phenotype of CD56^(dim/neg); andselecting for cells that have a phenotype of CD3+/CD 16+/CD 19+/CD52+.In addition, such methods can further include selecting for cells thathave a phenotype of CXCR4, CD123, HLADR, NKp30, NKp44, NKp46, CD11c, andCD162, and such methods can still further include selecting for cellsthat have a phenotype of, without limitation, CD11a, CD11b, CD62L, andFoxP3. A cellular composition produced by such methods also can includeCD34+ hematopoietic stem cells (HSCs).

Cells can be removed and/or selected for using an antibody (e.g., anantibody conjugated to a magnetic bead). In addition or alternatively,the hematopoietic cell composition can be separated by density gradientcentrifugation to obtain cells in the mononuclear cell fraction. In oneembodiment, the hematopoietic cell composition is contacted with agrowth factor. The hematopoietic cell composition can be obtained frombone marrow, thymus, peripheral blood, fetal liver, or embryonic yolksac. In one embodiment, cellular composition includes at least about 50%hFCs (e.g., at least about 75% hFCs).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this technology belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present methods and compositions, suitablemethods and materials are described below. In addition, the materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, and otherreferences mentioned herein are incorporated by reference in theirentirety.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages will be apparent from the drawings and detaileddescription, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 are graphs showing representative phenotypic analysis of hFCs.

FIG. 2 is a graph showing the CD56^(dim/neg) and CD56^(bright)sub-populations within CD8+/alpha beta TCR− hFCs.

FIG. 3A is a graph showing representative phenotypic analysis of theCD8+/alpha beta TCR−/CD56^(dim/neg) hFC sub-population, and FIG. 3B is agraph showing representative phenotypic analysis of the CD8+/alpha betaTCR−/CD56^(bright) hFC sub-population.

FIG. 4 shows the gating strategy for sorting and enumeration of humanHSCs.

FIG. 5 shows the gating strategy for sorting and enumerating human hFCs.

FIG. 6A is a graph showing the results of immunological monitoring inresponse to a number of stimulators 1-month post-transplantation intransplant patient SCD#3, and FIG. 6B is a graph showing the absoluteneutrophil count (ANC) in transplant patient SCD#4.

FIG. 7A is a graph showing the chimerism of transplant patient SCD#3,and FIG. 7B is a graph showing the source of hemoglobin from the sametransplant patient.

FIG. 8 are graphs showing the chimerism (Panel A), the source ofhemoglobin (Panel B), and the reticulocyte counts (Panel C) fortransplant patient SCD#4.

FIG. 9 are graphs showing the amount of the ANC and white blood cells(WBCs) (Panel A), the platelet count (Panel B), and the percentchimerism (Panel C) in a patient following solid organ transplant.

FIG. 10A is a graph showing the platelet count and FIG. 10B is a graphshowing the percent chimerism in patient #2 following solid organtransplant.

FIG. 11 are graphs showing the ANC (Panel A), the recovery of B cells,CD4+ cells, and CD8+ cells (Panel B), and the platelet count (Panel C)following solid organ transplant in patient #12.

FIG. 12 is a schematic showing a general nonmyeloablative conditioningand post-transplant immunosuppression regimen as exemplified herein.

FIG. 13 are graphs showing the chimerism (Panel A), multilineagechimerism (Panel B), response to a number of stimulators (Panel C),creatinine levels (Panel D), platelet count (Panel E), and white bloodcount and ANC (Panel F) in living donor kidney transplant Subject #3.

DETAILED DESCRIPTION

Human hFCs (hFCs) are provided that facilitate initial stem cellengraftment and that are required for sustained HSC engraftment. Alsoprovided are methods of purifying such hFCs from bone marrow or otherphysiological sources of hematopoietic cells. A variety of separationprocedures are provided, which generally are based on the presence orabsence of specific markers as disclosed herein.

Human Facilitating Cells (hFCs), Cellular Compositions Containing hFCs,and Methods of Making

Human facilitating cells (hFCs) have been identified and are describedherein. hFCs are generally characterized as CD8+ and alpha beta TCR−.hFCs also can be gamma delta TCR− or gamma delta TCR+(i.e., the absenceof gamma delta TCR cells is not required). The CD8+/alpha beta TCR− hFCscan be characterized by the presence of cells expressing the followingmarkers: CD3 epsilon (expressed by about 48% of hFCs), CD19 (expressedby about 33% of hFCs), CD11c (expressed by about 44% of hFCs), CD11b(expressed by about 40% of hFCs), Foxp3 (expressed by about 42% ofhFCs), HLA-DR (expressed by about 30% of hFCs), and CD123 (expressed byabout 8% of hFCs) (FIG. 1A). hFCs also can be characterized by thepresence of cells expressing IFN-gamma (about 25% of hFCs) and CXCR4(about 31% of hFCs) (FIG. 1B). In addition, about 65% of hFCs resembletolerogenic plasmacytoid dendritic cells (B220+/CD11c+/CD11b−), and hFCsare capable of inducing antigen-specific T_(reg) cells. Further, hFCscan be characterized by the presence, in lower levels, of markers suchas, without limitation, CD16, CD52, NKp30, NKp44, NKp46, CD162, CD11aand CD62L.

Within the population of CD8+/alpha beta TCR− hFCs, there are twosubpopulations: CD8+/alpha beta TCR−/CD56^(dim/neg) (about 55% of hFCs)and CD8+/alpha beta TCR−/CD56^(bright) (about 45% of hFCs) (FIG. 2). Asis understood by those skilled in this art, CD56^(dim/neg) cells referto a population of cells that express a relatively small amount of CD56(CD56^(dim)) and cells that do not express CD56 (CD56^(neg)); whileCD56^(bright) cells refer to cells that express a relatively largeamount of CD56 (CD56^(bright)).

Within the CD8+/alpha beta TCR−/CD56^(dim/neg) sub-population of hFCs,the majority of cells express CD3 epsilon (about 80%), approximately athird of the cells express HLA-DR (about 30%), and a lower percentage ofcells express CD11c (about 17%), CD19 (about 16%), CD11b (about 14%),and CD123 (about 11%) (FIG. 3A). Thus, the majority of cells within theCD8+/alpha beta TCR−/CD56^(dim/neg) hFC sub-population are CD3epsilon+/CD19−. Within the CD8+/alpha beta TCR−/CD56^(bright)sub-population of hFCs, approximately 65% of cells express CD11c, about67% of the cells express CD11b, and about 40% of the cells expressHLA-DR, while CD3 epsilon, CD19, and CD123 are expressed at much lowerlevels (about 29%, about 25%, and about 10%, respectively) in thissub-population (FIG. 3B). Thus, the majority of cells within theCD8+/alpha beta TCR−/CD56^(bright) hFC sub-population are CD3epsilon−/CD19+.

hFCs can be obtained from bone marrow, or any other physiologic sourceof hematopoietic cells such as, without limitation, the spleen, thymus,blood, embryonic yolk sac, or fetal liver. In one embodiment, hFCs areobtained from mobilized peripheral blood (in the presence of, forexample, granulocyte colony-stimulating factor (G-CSF) orgranulocyte-macrophage colony stimulating factor (GM-CSF). In anotherembodiment, hFCs are obtained from vertebral bone marrow.

Once hematopoietic cells are obtained, hFCs can be enriched, purified(or substantially purified) by various methods that typically useantibodies that specifically bind particular markers to select thosecells possessing (or lacking) those particular markers. Cell separationtechniques include, for example, cell sorting using a fluorescenceactivated cell sorter (FACS) and specific fluorochromes; biotin-avidinor biotin-streptavidin separations using biotin conjugated to cellsurface marker-specific antibodies and avidin or streptavidin bound to asolid support (e.g., affinity column matrix or a plastic surface);magnetic separations using antibody-coated magnetic beads; ordestructive separations such as antibody and complement or antibodybound to cytotoxins or radioactive isotopes. Methods of makingantibodies that can be used in cell separations are well known in theart. See, for example, U.S. Pat. No. 6,013,519.

Separation using antibodies directed toward specific markers can bebased upon negative or positive selections. In separations based onnegative selection, antibodies that are specific for markers that arepresent on undesired cells (non-hFCs) and that are not present on thedesired cells (hFCs) are used. Those (undesired) cells bound by theantibody are removed or lysed and the unbound cells retained. Inseparations based on positive selection, antibodies that are specificfor markers that are present on the desired cells (hFCs) are used. Thosecells bound by the antibody are retained. It will be understood thatpositive and negative selection separations may be used concurrently orsequentially. It will also be understood that the present disclosureencompasses any separation technique that can be used to enrich orpurify the hFCs described herein.

One well-known technique for antibody-based separation is cell sortingusing, for example, a FACS. Briefly, a suspended mixture ofhematopoietic cells are centrifuged and resuspended in media. Antibodiesthat are conjugated to fluorochromes are added to allow the binding ofthe antibodies to the specific cell surface markers. The cell mixture isthen washed and run through a FACS, which separates the cells based ontheir fluorescence, which is dictated by the specific antibody-markerbinding. Separation techniques other than cell sorting additionally oralternatively can be used to obtain hFCs. One such method isbiotin-avidin (or streptavidin)-based separation using affinitychromatography. Typically, such a technique is performed by incubatinghematopoietic cells with biotin-coupled antibodies that bind to specificmarkers, followed by passage of the cells through an avidin column.Biotin-antibody-cell complexes bind to the column via the biotin-avidininteraction, while non-complexed cells pass through. The column-boundcells can be released by perturbation or other known methods. Thespecificity of the biotin-avidin system is well-suited for rapidseparation.

Cell sorting and biotin-avidin techniques provide highly specific meansfor cell separation. If desired, less specific separations can beutilized to remove portions of non-hFCs from the hematopoietic cellsource. For example, magnetic bead separations can be used to initiallyremove non-facilitating differentiated hematopoietic cell populationsincluding, but not limited to, T-cells, B-cells, natural killer (NK)cells, and macrophages (MAC) as well as minor cell populations includingmegakaryocytes, mast cells, eosinophils, and basophils. In addition,cells can be separated using density-gradient separation. Briefly,hematopoietic cells can be placed in a density gradient prepared with,for example, Ficoll or Percoll or Eurocollins media. The separation canthen be performed by centrifugation or automatically with, for example,a Cobel & Cell Separator '2991 (Cobev, Lakewood, Colo.). Additionalseparation procedures may be desirable depending on the source of thehematopoietic cell mixture and its content. For example, if blood isused as a source of hematopoietic cells, it may be desirable to lyse redblood cells prior to the separation of any fraction.

Although separations based on specific markers are disclosed, it will beunderstood that the present disclosure encompasses any separationtechnique(s) that result in a cellular composition that is enriched forhFCs, whether that separation is a negative separation, a positiveseparation, or a combination of negative and positive separations, andwhether that separation uses cell sorting or some other technique, suchas, for example, antibody plus complement treatment, column separations,panning, biotin-avidin technology, density gradient centrifugation, orother techniques known to those skilled in the art. Most sources ofhematopoietic cells naturally contain about 0.5% to about 8% (e.g.,typically about 1%) hFCs. The separations such as those disclosed hereincan yield cellular compositions that are enriched for hFCs (i.e.,include a greater number of hFCs than are found naturally inphysiological hematopoietic cell sources). For example, cellularcompositions are provided in which at least about 5% (e.g., at leastabout 8%, 10%, 12%, 15%, 20% or more) of the cells are hFCs as describedherein. These compositions are referred to as “enriched” for hFCs. Inanother example, cellular composition are provided in which at leastabout 30% (e.g., at least about 35%, 40%, 50% or more) of the cells arehFCs as described herein. The compositions are referred to as “purified”for hFCs. Further processing, by either or both positive or negativeselections, can yield cellular compositions in which at least about 60%of the cells (e.g., at least about 70%, 75%, 80%, 85%, 90%, 95%, or 99%)are hFCs as described herein.

Exemplary methods of obtaining cellular compositions that include hFCsare described herein. Those skilled in this art would understand thatthe examples described herein can be modified in a number of ways tostill obtain hFCs or to obtain different amounts of hFCs. In thefollowing examples, bone marrow is the source of hematopoietic stemcells. Bone marrow can be harvested (e.g., from a donor) by variousmethods well known to those skilled in the art. For example, bone marrowcan be harvested from the long bones (e.g., femora or tibia), but alsocan be obtained from other bone cavities or the spine.

In one exemplary method, non-hFCs and non-HSCs can be removed from thebone marrow using one or more negative selections described herein. Forexample, T cells, also known as graft vs. host disease (GVHD)-producingcells, can be specifically removed from the cellular composition usingantibodies directed toward T cell-specific markers such as alpha betaTCR+. In certain embodiments, an antibody directed toward delta gammaTCR+ can be used to remove a further subset of T cells. The resultingcellular composition is enriched for hFCs and HSCs, and also willcontain other immature progenitor cells such as immature lymphoid andmyeloid progenitor cells. Representative cellular compositions enrichedfor hFCs were deposited with the American Type Culture Collection (ATCC:Manassas, Va.) on ______ and assigned Accession Nos. ______. Thesedeposits were made for reference purposes only and were not made forpurposes related to 35 U.S.C. §112.

In another exemplary method, hFCs can be obtained from the bone marrowusing one or more positive selections described herein. For example,hFCs can be purified by cell sorting (e.g., using FACS) with one or moreof the markers described herein (e.g., CD8+, CD19, CD56).

In certain instances (e.g., non-therapeutic), it may be desirable toremove the HSCs from the cellular composition. HSCs can be removed frombone marrow using, for example, antibodies that bind CD34+ and,optionally, CD45+. See, for example, U.S. Pat. No. 5,061,620 or the LCLaboratory Cell Separation System, CD34 Kit (CellPro, Inc., Bothell,Wash.).

Methods of Using hFCs and Cellular Compositions Containing hFCs

The ability of hFCs to enhance engraftment of donor bone marrow cells ina recipient indicates that hFCs are useful in facilitating varioustherapy protocols. Using a cellular composition that is enriched forhFCs (e.g., contains about 5% to about 12% hFCs) significantly improvesdurable engraftment and eliminates graft vs. host disease (GVHD).Although not bound by any particular mechanism, it is believed that,once administered, the hFCs home to various hematopoietic cell sites inthe recipient's body, including bone cavity, spleen, fetal or adultliver, and thymus. The hFCs become seeded at the proper sites, engraft,and begin establishing a chimeric immune system. It is possible thatboth the stem cells and the hFCs complex together to seed theappropriate site for engraftment.

Methods of administering a therapeutic cellular composition comprisinghFCs to a recipient also are described herein. A therapeutic cellularcomposition as used herein refers to a composition that includes hFCsand HSCs. Such a composition can be produced using any of the methodsdescribed herein (e.g., positive and/or negative selections). Atherapeutic cellular composition for administration to a recipient mayinclude a total of between about 1×10⁸ cells and 3×10⁸ cells perkilogram of dosing weight of the recipient. Within a therapeuticcellular composition, the number of HSCs can be between about 1×10⁵ and18×10⁶ HSCs per kg of recipient dosing weight, and a similar range ofhFCs can be administered. The exact numbers of cells that are used,however, will depend on many factors, including the number of cells inthe original source of hematopoietic stem cells, the number of cells(e.g., hFCs and/or HSC) present after processing (e.g., enrichmentand/or purification), as well as the condition of the recipient'shealth.

As described herein, obtaining hFCs typically involves depleting thealpha beta TCR+ T cells, as these are considered GVHD-producing cells.Therapeutically, however, the presence of alpha beta TCR+ T cells hasbeen found to be beneficial in the cellular composition of HSCs andhFCs. As shown in the Example section herein, a cellular compositionthat includes alpha beta TCR+ T cells at a level that is greater than isgenerally considered to be therapeutic surprisingly promoted chimerismand engraftment. It is generally accepted that about 1×10⁵ alpha betaTCR+ T cells/kg of recipient body weight is considered a lethal amountof T cells. However, in the methods described herein, amounts greaterthan that were routinely administered to recipients without adverseeffects. Specifically, amounts between about 2.0×10⁶ and 5.0×10⁶ alphabeta TCR+ T cells (e.g., between about 2.5×10⁶ and 4.5×10⁶ alpha betaTCR+ T cells/kg recipient body weight; between about 3.0×10⁶ and 4.0×10⁶alpha beta TCR+ T cells/kg recipient body weight; about 3.0×10⁶ and4.2×10⁶ alpha beta TCR+ T cells/kg recipient body weight; about 3.2×10⁶alpha beta TCR+T cells/kg recipient body weight; or about 3.8×10⁶ alphabeta TCR+ T cells/kg recipient body weight) can be included in atherapeutic cellular composition.

Accordingly, depending on the procedures and methods used to obtain theHSC and hFC therapeutic cellular composition, the number of alpha betaTCR+ T cells in the composition may need to be adjusted. For example, incertain embodiments, alpha beta TCR+ T cells can be added back to the Tcell-depleted HSC and hFC composition in order to obtain the desirednumber. In other embodiments, the depletion step can be modified so thatonly the desired about of T cells are depleted, thereby leaving thedesired amount of T cells in the composition. In order to achieve thedesired amount of T cells in a therapeutic cellular composition, thenumber of T cells can be determined, for example, prior to depletion(e.g., in the starting material) or following the depletion step.Methods of determining the number of cells (e.g., T cells) in a sampleare well known in the art (e.g., FACS).

Therapeutic cellular compositions generally are administeredintravenously, but other modes of administration such as direct boneinjection can be used. The therapeutic cellular compositions describedherein, even in the presence of higher-than-therapeutic levels of alphabeta TCR+ T cells, result in durable chimerism. As used herein, durablechimerism refers to a recipient's immune system that is at least about1% (e.g., at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 25%, 50%,75% or more (e.g., 100%)) donor origin for greater than 6-monthspost-transplant (e.g., 1-year or more post-transplant). In addition,durable chimerism can be achieved using the therapeutic cellularcomposition described herein even in recipients who are not HLA-matchedto their donor or who are only partially matched with their donor.Accordingly, the therapeutic cellular compositions described hereinallow for transplantation between a donor and a recipient that aresyngeneic to one another and should allow for transplantation between adonor and a recipient that are allogeneic to one another.

Traditionally, methods of establishing a chimeric immune system requireddestroying the immune system of the recipient, which results in ablationof the recipient's HSCs. This may be accomplished by techniques wellknown to those skilled in the art and include, without limitation,irradiating the recipient with selected levels of total bodyirradiation, administering specific toxins or chemotherapeutic agents tothe recipient, administering specific monoclonal antibodies ormonoclonal antibodies attached to toxins or radioactive isotopes to therecipient, or combinations thereof. Notably, administering the hFCsdescribed herein (e.g., in the therapeutic cellular composition) to arecipient significantly reduces the amount conditioning required of arecipient for successful engraftment and also significantly reduces theamount of immunosuppression required following transplantation. Forexample, destroying a recipient's immune system often involves lethallyirradiating the recipient with 950 centigray (cGy) of total bodyirradiation (TBI), while the procedures described herein utilize aconditioning regimen with as little as 25 cGy to 200 cGy of TBI.

The ability to establish successful chimerism allows for significantlyimproved survival following transplant. The present disclosure providesfor methods of transplanting a donor physiological component, such as,for example, organs, tissue, or cells. Using the hFCs in the methodsdisclosed herein results in a recipient who has a chimeric immunesystem, which is completely immunotolerant to transplanted donor organ,tissue, or cells, but competently rejects third party grafts.Transplanted donor organ, tissue, or cells are able to perform theirrespective functions in the recipient. For example, transplanted isletcells can provide an effective treatment for diabetes. In addition,permanent acceptance of endocrine tissue grafts (thyroid, parathyroid,adrenal cortex, adrenal medulla, islets) as well as kidney, liver,heart, and composite tissues such as face, hand and other extremitieshas been demonstrated. It will be understood that a mixed chimericimmune system can be produced in a recipient before, during, or aftertransplantation of an organ, tissue or cells, but typically is producedbefore or at the same time as the transplantation.

The use of hFCs in establishing a chimeric immune system cansignificantly expand the scope of diseases that can be treated usingbone marrow transplantation. Beyond transplantation (e.g., heart,kidney, liver, pancreatic islets, and hand or face), the ability toestablish a successful chimeric hematopoietic system in a recipient canbe used to treat other diseases or disorders that are not currentlytreated by bone marrow transplantation because of the morbidity andmortality associated with GHVD. Autoimmune diseases involve attack of anorgan or tissue by one's own immune system. However, when a chimericimmune system is established, the body can relearn what is foreign andwhat is self Establishing a chimeric immune system using the hFCsdescribed herein can reduce or halt the autoimmune attack causing thecondition. Autoimmune diseases that can be treated using the hFCsdescribed herein include, for example, type I diabetes, systemic lupuserythematosus, multiple sclerosis, rheumatoid arthritis, psoriasis, orCrohn's colitis.

It may also be possible to treat Alzheimer's disease using the cellularcompositions described herein. The cellular compositions disclosedherein also can be used to treat hemoglobinopathies such as, forexample, sickle cell anemia, spherocytosis or thalassemia, as well asmetabolic disorders such as Hunters disease, Hurlers disease, chronicgranulomatous disease, leukodystrophy, and enzyme defects. In addition,the cellular compositions described herein can be used to treatleukemias or other rare childhood disorders (e.g., ADA deficiency,aplastic anemia or SCID), or the cellular compositions described hereincan be used in regenerative repair (e.g., macular degeneration,myocardial infarction, or islet regeneration).

In accordance with the present disclosure, there may be employedconventional molecular biology, cell biology, microbiology andbiochemical techniques within the skill of the art. Such techniques areexplained fully in the literature. The methods and compositions will befurther described in the following examples, which do not limit thescope of the methods and compositions described in the claims.

EXAMPLES Section A Colony Forming Cell Assays Example 1 Purification ofHSC and hFC

HSC and hFC were isolated from Human Vertebral Bone Marrow (VBM) orMobilized Peripheral Blood (MPB) by multiparameter, live sterile cellsorting (FACSVantage SE: Becton Dickinson). Briefly, VBM or MPB wasstained with directly labeled monoclonal antibodies (mAbs) at saturatingconcentrations for 30 min. HSCs: CD34+/CD45+; and hFCs:CD8+/TCR−/CD56^(dim/neg). Both cell populations were sorted and analyzedfor purity. Only 85% or greater purity levels were accepted.

Example 2 HSC and hFC Sorting and Enumeration

HSCs were sorted and enumerated based on the ISHAGE protocol. See,Sutherland et al., 1996, “The ISHAGE guidelines for CD34+ celldetermination by flow cytometry,” J. Hematotherapy, 5:213-26. Briefly,CD45−FITC/CD34−PE combination parameters provided a clinically relevantreflection of the peripheral blood stem/progenitor cell compartment.Plot 1 was formatted with Forward Scatter (FSC; x-axis) vs Side Scatter(SSC; y-axis), and a region (R1) was drawn around the lymphocyte,monocyte and granulocyte populations excluding debris. From R1, Plot 2was formatted with CD45 FITC versus Side Scatter, and R1 was drawn sothat CD45− events were excluded. From R2, Plot 3 was formatted with CD34PE versus Side Scatter, and R3 was drawn only around the CD34+population. From R3, Plot 4 was formatted with CD45−FITC versus SSC ofCD34+ cells. Cells forming a cluster with characteristic low SSC and lowto intermediate CD45 fluorescence were gated and designated R4.Nonspecific stained events were excluded from this region. From R4, Plot5 was formatted with FSC (x-axis) versus SSC (y-axis). A cluster ofevents meeting all the fluorescence and light scatter criteria of CD34+stem/progenitor cells appeared in Plot 5.

FIG. 4 shows the gating strategy for the sorting and enumeration ofhuman HSCs using the ISHAGE protocol. FIG. 5 shows the gating strategyfor the sorting and enumeration of human hFCs.

Example 3 Colony-Forming Cell Assay with hFCs

Following cell sorting, HSCs (CD45+/CD34+) alone or HSCs and hFCs(CD45+/CD34+ plus CD8+/TCR−/CD56^(dim/neg)) or HSCs+T cells as a controlwere either immediately plated in methylcellulose (0 hr) orpre-incubated for 18 hrs in cell culture media before plating inmethylcellulose. All cell samples were cultured in quadruplicate. After14 days of culturing at 37° C. and 5% CO₂, colonies containing more than50 cells were scored.

Without pre-incubation, there was no significant difference in coloniesgenerated by HSC alone vs. HSC plus hFC. Strikingly, when HSCs wereco-incubated with hFCs for 18 hrs prior to placement in the CFC assay,hFC significantly (p<0.005) enhanced colony formation compared to HSCalone and HSC co-incubated with CD8+ T cells. These results indicatethat human hFCs, like mouse hFCs, exert a protective effect on HSCs andpromote the generation of more primitive multipotent progenitors invitro.

Example 4 Colony-Forming Cell Assay with a Sub-Population of hFCs

Colony Forming Culture (CFC) Assay: 15,000 HSCs were cultured with orwithout 30,000 CD8+/alpha beta TCR−/CD56^(dim/neg) hFCs for 0 hrs or 18hrs in culture media in a 96 well plate and incubated at 37° C. Afterculture, cells were resuspended in methylcellulose and used in a CFCAssay. Colonies were counted on day 14.

Summary and Results: To evaluate the function of CD8+/alpha betaTCR−/CD56^(dim/neg) hFCs in vitro, HSCs were incubated with CD8+/alphabeta TCR−/CD56^(dim/neg) hFCs for 18 hrs and then cultured inmethylcellulose for 14 days in a colony-forming cell assay. HSC plusCD8+/alpha beta TCR−/CD56^(dim/neg) hFCs generated significantly morecolonies compared with HSCs alone (p=0.0038), demonstrating thatCD8+/alpha beta TCR−/CD56^(dim/neg) hFCs have a direct effect on theclonogenicity of HSCs.

Section B Characterization of Human hFCs In Vivo Example 1 Chimerism andEngraftment in a Mouse Model

It has been shown previously that CD8⁺/TCR⁻ hFCs enhance engraftment ofpurified HSCs in allogeneic and syngeneic mouse recipients (Fugier etal., 2005, J. Exp. Med., 201(3):373-383). In addition, it has been shownin mice that hFCs enhance the clonogenicity and promote the generationof more primitive multipotent HSC progenitors in vitro (Rezzoug et al.,2008, J. Immunology, 180(1):49-57).

One goal was to achieve human HSC chimerism in a mouse model. Briefly,CD34⁺, CD45⁺ human HSCs were sorted from G-CSF mobilized peripheralblood, and 100,000 sorted human HSCs were transplanted into NOD/SCID/IL2receptor (IL2R) γ chain^(null) mice conditioned with 325 cGy TBI. Wholeblood was collected from transplanted mice one month followingtransplantation, and PBL typing was performed using antibodies specificfor human T cells, B cells, natural killer cells, dendritic cells, andmonocytes. Results showed that an average of 3.2% human HSC chimerismwas achieved following transplantation with 100,000 hHSCs.

Experiments then were performed in which 100,000 hHSCs alone or 100,000hHSCs+300,000 hFCs were transplanted into NOD/SCID/IL2Rγ^(null) miceconditioned with 325 cGy TBI. Multilineage PBL typing was performed at30 days after transplantation as described above.

The results of these experiments demonstrated that the HSC+hFC groupproduced a higher percentage of human T cells (CD4, CD8, DC; seeTable 1) and human monocytes (CD33; Table 2) compared to the HSC alonegroup. The percentage of donor chimerism in lymphoid gate and myeloidgate are summarized in Table 3.

TABLE 1 Percentage of human T cells, NK cells, B cells, and DCs inlymphoid gate T cells αβ/δγ NK B cell DC Group Mouse CD8 CD4 CD3 TCRCD56 CD19 CD11c HSC A 0 0.1 0.1 0.1 0.1 0.1 0 alone B 0 0 0.1 0.3 0.2 00 C 0 0 0 0.1 0 0 0 HSC + D 0.1 0.5 0.4 0.5 0.1 0.1 0.1 hFC E 0.1 0.10.1 0.1 0.1 0 0.1 F 0 0.1 0.1 0.1 0.1 0.1 0.1

TABLE 2 Percentage of human DCs and monocytes in myeloid gate GroupMouse CD11c CD33 HSC alone A 4.8 9.2 B 0.6 5 C 0 0 HSC + hFC D 3.2 6.6 E4.1 8.1 F 8.2 14.9

TABLE 3 Percentage of human hematopoietic cells Lymphoid Myeloid GroupMouse CD45 CD45 HSC alone A 1.5 9.2 B 1.5 5.8 C 0.3 0.3 HSC + hFC D 1.18.2 E 0.8 9.4 F 1.2 15.9

Example 2 Engraftment of CD8+/Alpha Beta TCR−/CD56^(Dim/Neg) in a MouseModel

Animals: Five to 6-week-old male non-obese diabetic(NOD)/SCID/interleukin-2 receptor (IL-2r) gamma-chain knockout (NSG)mice were purchased from the Jackson Laboratory (Bar Harbor, Me.).

Purification of HSCs and hFCs: HSCs and FCs were sorted from humanG-CSF-mobilized peripheral blood by multiparamter, live sterile cellsorting (FACSVantage SE and FACSAria; Becton Dickinson, Mountain View,Calif.).

Phenotype of human CD8+/alpha beta TCR− hFCs: G-CSF mobilized PBMC werestained with anti-human CD8 alpha, alpha beta TCR, delta gamma TCR,CD56, CD3 epsilon, CD19, CD11c, CD11b, HLA-DR, Foxp3, INF-gamma,TGF-beta, CXCR4, and SDF-1 monoclonal antibodies, and analyzed by LSRusing Cell Quest Software (Becton Dickinson).

HSC and FC transplantation: In the human HSC+FC xenogeneic model,100,000 human HSCs with or without 300,000 sorted CD8+/alpha betaTCR−/CD56^(dim/neg) hFCs were transplanted into NOD/SCID/IL-2rgamma^(null) mice recipients conditioned with 325 cGy TBI.

Assessment of chimerism: Donor cell engraftment was evaluated inperipheral blood lymphocytes, bone marrow cells and splenocytes using7-color flow cytometry.

Summary: To evaluate whether human CD8+/alpha beta TCR−/CD56^(dim/net)hFCs enhance engraftment of human HSCs in vivo, 100,000 HSC alone orplus 300,000 CD8+/alpha beta TCR−/CD56^(dim/net) hFCs was transplantedinto NOD/SCID/IL2rg^(null) (NSG) recipient mice conditioned with 325 cGyof total body irradiation. At 30 days after transplantation, 8 of 21(38%) recipients of HSC alone engrafted. In contrast, 81% of recipients(n=16) receiving HSC plus CD8+/alpha beta TCR−/CD56^(dim/net) hFCsengrafted, and donor lymphocyte and donor monocyte chimerism inperipheral blood was 0.53%±0.16% and 3.93%±1.28%, respectively.

At 6 months after transplantation, NSG recipients of HSC alone lostdonor chimerism in peripheral blood and little to no donor cells weredetected in spleen and bone marrow. In contrast, NSG recipients ofHSC+CD8+/alpha beta TCR−/CD56^(dim/net) hFCs exhibited durable donorchimerism in peripheral blood and showed significantly higher levels ofdonor chimerism in spleen (about three-times as many donor lymphocytesand about twice as many donor monocytes) and bone marrow (aboutten-times as many donor lymphocytes and about four-times as many donormonocytes) compared to recipients of HSC alone.

Section C Treatment of Sickle Cell Disease (SCD) in Humans Example 1 TheSickle Cell Disease (SCD) Preliminary Experiment

Two sickle-cell disease (SCD) patients were previously treated in apilot experiment to try to establish mixed chimerism. Both SCD patientswere at high risk for complications from their disease. Whether acombination of 200 cGy TBI with fludarabine, MMF, and CyA couldestablish engraftment in patients with SCD was evaluated. Only transientengraftment, however, was achieved. The conditioning was well tolerated,and no severe adverse events occurred; however, endogenous hematopoiesisreappeared.

To overcome the transfusion/sensitization barrier, improvements weremade to the protocol. For example, Campath, which is a humanizedanti-CD52 monoclonal antibody that is a powerful lytic agent for matureT cells, B cells and NK cells, was added to the clinical conditioningregimen. Two cycles of Campath were administered (month −2 and month −1)with the rationale that the first cycle would deplete mature B cells andcause homeostatic proliferation of memory B cells to replace thedepleted B cells and the second cycle would deplete the proliferatingmemory B cells. It was hypothesized that the broad lymphoid specificityof Campath would provide a powerful approach to target T and B cells inthe recipient that mediate the alloreactivity induced by transfusiontherapy.

In one example, four doses of 10 mg/day of Campath-1H were administeredat day −53 to −50, and another four doses of 7 mg/day of Campath-1H wereadministered at day −24 to −21. 30 mg/m² of Fludarabine was administeredat day −5 to −3, and 200 cGy total body irradiation was administered atday −1 along with Mycophenolate mofetil and cyclosporine, which wascontinued until durable engraftment. FCs+HSCs were transplanted at day0.

Two subjects with SCD have been successfully transplanted under therevised protocol. Both subjects have maintained engraftment at 27 and 24months post-transplant and are asymptomatic and transfusion independent.It was demonstrated that mixed chimerism can be established with minimaltoxicity in sensitized recipients through partial recipient conditioningfollowed by transplantation with HSCs and hFCs to reduce the risk ofGVHD while preserving engraftment. The reduced-intensity conditioningapproach described herein is safe, well-tolerated and, in combinationwith the HSC+hFC graft, sufficient to induce stable mixed chimerism anddominantly normal RBC production in transfused patients.Immunocompetence to respond to PHA, Candida, and alloantigen returned by1 month post-transplant (FIG. 6A; a stimulation index of >3 is positive(horizontal line on graph)). The nadir occurred between day 9 and day 24for both patients (absolute neutrophil count [ANC]<1,000) (FIG. 6B).

Example 2 SCD Patient #3—Transplanted in November 2005

SCD#3 (Date of Birth Feb. 11, 1998) is an African American female whoexperienced multiple pain crises and episodes of acute chest syndrome.She was maintained on transfusion therapy. Her HLA-identical sister withsickle cell trait served as her donor. The patient was conditioned withfour doses of Campath-1H (30 mg/day) starting at day −53, and a secondround of four doses of Campath-1H (30 mg/day) starting at day −24. Shereceived 3 doses of fludarabine (30 mg/m² IV) starting at day −4, andthen 200 cGy of TBI on day 0.

Post-transplant, she was treated with cyclosporine (1.5 mg/kg/bid) andMMF for 22 months. The immunosuppression was subsequently tapered andhas been discontinued completely. The patient received 14.1×10⁶ CD34+cells/kg body weight, 43.5×10⁶ alpha beta TCR cells/kg body weight and5.4×10⁶ hFCs/kg body weight. She showed 5% donor cell chimerism on Day17 and 78% donor cell chimerism on day 32. She has been asymptomatic andhas not required transfusions post transplant. At day 727post-transplantation, she was 21% donor cell chimeric (FIG. 7A), and shehad no evidence of GVHD. Although her total donor chimerism wasapproximately 30%, she was producing nearly 100% donor-derived trait RBC(FIG. 7B). At 1259 days post-transplantation, she produced 100% donorRBC and had T, B, and myeloid chimerism ranging between 10-30%. She wasstill transfusion independent and had not had any complications from herSCD.

During the processing procedure for SCD #3, some difficulty wasexperienced in recovering the correct fraction in the cell separation(Percoll) procedure due to the density of SCD trait marrow cells.Because the donor/recipient pair were HLA matched, the decision was madeto abort the process and not deplete the product. Therefore, the patientreceived whole bone marrow. However, the efficacy of the conditioningwas established in this candidate.

Example 3 SCD Patient #4—Transplanted in March 2006

SCD#4 (Date of birth May 23, 1996) is a Nigerian male who sufferedmultiple pain crises and two acute chest syndromes prior to starting redcell exchange in 1999. The patient's HLA-identical sibling who hadsickle cell trait served as his donor. The patient received the samenon-myeloablative conditioning as SCD#3. His HSC+hFC dose was5.24×10⁶/kg CD34 cells, 0.55×10⁶/kg αβ-TCR cells and 0.35×10⁶/kg hFC. Hetolerated the conditioning very well and engrafted and chimeric (88%donor cells) at one month based on FISH. His donor chimerism was 28% asof day 697 (FIG. 8A). Donor T cell chimerism was 34% at day 501. Thepatient has remained asymptomatic since his transplant and is producingpredominantly normal RBC (FIG. 8B). The reticulocyte counts for patientSCD#4 has ranged between 0.5% and 1%, which is within normal ranges(FIG. 8C).

Example 4 Summary

This section described successful transplantation of twoheavily-transfused SCD patients using HLA-identical marrow from siblingdonors. Both patients were successfully transplanted usingreduced-intensity non-myeloablative conditioning and have remaineddisease free for >2 years. At enrollment, they weretransfusion-dependent and at very high risk for painful crises and othercomplications. Both patients have been successfully weaned fromimmunosuppression.

Section D Treatment of Sickle Cell Disease in Humans

Five individuals at high risk for morbidity and mortality from theirthalassemia were enrolled on the protocol according to the inclusion andexclusion criteria below.

Example 1 Inclusion Criteria

The following criteria were established to identify individuals withthalassemia who have a high predicted morbidity and are at risk forearly mortality: patients with alpha or beta thalassemia major; orpatients with other complex and transfusion-dependenthemoglobinopathies. Individuals must also meet all of the followinggeneral inclusion criteria: individuals must have a related donor(identical or mismatched for 1, 2 or 3 HLA-A, -B or -DR loci);individuals must have adequate cardiopulmonary function as documented byechocardiogram or radionuclide scan (shortening fraction >26% orejection fraction >40% or >80% of normal value for age); individualsmust have adequate pulmonary function documented by FEV1 of ≧50% ofpredicted for age and/or DLCO (corrected for hemoglobin)≧50% ofpredicted for age for patients >10 years of age (if patient cannotperform PFT's, resting pulse oximeter >85% on room air or clearance bythe pediatric or adult pulmonologist is required); individuals must haveadequate hepatic function as demonstrated by a serum albumin >3.0 mg/dL,and SGPT or SGOT <5 times the upper limit of normal; and individualsmust have adequate renal function as demonstrated by a serum creatinine<2 mg/dL. If serum creatinine is >2 mg/dL, then a creatinine clearancetest or nuclear medicine GFR should document GFR of ≧50 ml/min/1.73 m².There are no age limits for this protocol.

Example 2 Exclusion Criteria

Individuals are excluded from this trial if they meet any of thefollowing criteria: the individual lacks related donors; the individualhas uncontrolled infection or severe concomitant diseases, and may nottolerate reduced intensity transplantation; the individual exhibitssevere impairment of functional performance as evidenced by a Karnofsky(patients >16 years old) or Lansky (children <16 years old) score of<70%; the individual exhibits renal insufficiency (GFR <50 ml/min/1.73m²); the individual has a positive human immunodeficiency virus (HIV)antibody test result; the individual is pregnant as indicated by apositive serum HCG test; the individual's only donor is pregnant at thetime of intended transplant; the individual is of childbearing potentialand is not practicing adequate contraception; the individual has beenexposed to previous radiation therapy that would preclude TBI; theindividual is a Jehovah's witness; the individual has uncontrolledhypersplenism; or the individual exhibits severe alloimmunization withinability to guarantee a supply of adequate PRBC donors.

Example 3 Recipient Evaluation

A complete history and physical examination of the individual isperformed. Estimation of pre-HSCT Lansky or Karnofsky status isobtained. The history includes: age of diagnosis, overall growth anddevelopment, frequency and number of transfusions, any aplastic crises,prior treatment (e.g., hydroxyurea), baseline HbF plus A2 levels,alloimmunization status, treatment and dates, any MRI scans, transfusiontherapy, infections, aseptic necrosis, history of hepatitis, ironoverload, prior liver biopsies, and pathologic findings.

The following hematological tests are performed: CBC (Hgb, Hct, MCV,MCHC, RDW, platelet, white blood cell count), differential count,reticulocyte count, ferritin, folate, quantitative Hgb electrophoresis,PT, PTT, fibrinogen, direct and indirect Coombs test. In addition, thealpha gene number is determined, the beta-globin haplotype isdetermined, globin chain synthetic studies are performed, and thesubject is ABO Rh typed and screened.

The following chemistries are obtained: total and direct bilirubin,SGPT, SGOT, alkaline phosphatase, Protein C, IgG subclasses, albumin,Ca++/PO4++/Mg++, serum electrolytes, BUN/creatinine, urinalysis,creatinine clearance/GFR; and endocrine levels of T4, TSH, FSH, LH, andgrowth hormone.

The individual is HLA typed (HLA A, B, C, DQ and DR typing) based onmolecular analysis.

The following diagnostic tests are performed on the individual: a CTscan (brain, sinuses, chest, abdomen, pelvis), PFTs (crying vitalcapacity for younger children unable to perform conventional PFT, DLCOfor patients >10 years), EKG, echocardiogram or MUGA scan, liver andspleen scan, ultrasound of gall bladder, bone age, and estradiol ortestosterone.

The individual is screened for the following infectious markers: CMV,IgG, PCR, HSV & VZV IgGs, HIV 1 and 2 antibody and PCR, HTLV 1 and 2antibody, Hepatitis B surface antigen, Hepatitis B core antibody,Hepatitis C antibody and PCR, EBV IgG and IgM, toxoplasma IgG and IgM,West Nile Virus NAT, Trypanosoma cruzi (Chagas) antibody, RPR orequivalent.

Example 4 Donor Evaluation and Selection

HLA-identical donor and recipients are used, or donor and recipientmismatched pairs (e.g., up to haploidentical (parent, aunt, uncle,cousin, or sibling)) are used. Family members willing to donate bonemarrow are HLA-typed. The best available match is selected. All donorsparticipating are evaluated as per FDA regulations for donor screeningprior to stem cell harvest. All evaluations are completed within 30 daysof the transplant. Pediatric donors are considered for mobilization. Ifthe donor is not a good candidate for apheresis, bone marrow isharvested from the iliac crest. If more than one related donor isavailable, the closer matching, younger, and/or CMV-negative donor isselected. All donors are placed on iron replacement therapy. Phereseddonors can be supplemented with Vitamin K and/or calcium.

Donors are screened as described herein and the following information isobtained. The history and physical examination of the donor is obtainedincluding pregnancy and transfusion history. Donors are screened forCBC, differential; PT with INR, PTT and fibrinogen; ABO and Rh Type andscreen, ferritin, iron and TIBC; HLA typing: HLA class I (-A, -B, -C)and class II (-DR, -DQ) typing by molecular analysis; hemoglobinelectrophoresis (thalassemia trait is acceptable); SGPT or SGOT,alkaline phosphatase, and bilirubin (total and direct); serum pregnancytest; serum electrolytes, BUN, and creatinine; CMV, IgG, PCR, HSV & VZVIgG, HIV 1 and 2 antibodies and PCR, HTLV 1 and 2 antibodies, HepatitisB surface antigen, Hepatitis B core antibody, Hepatitis C antibody andPCR, EBV IgG and IgM, Toxoplasma IgG and IgM, West Nile Virus NAT,Trypanosoma cruzi (Chagas), RPR or equivalent test; hepatitis B coreantibody (if antibody-positive, perform PCR for viral DNA, accept donorif negative); hepatitis B surface antigen (reject Hepatitis B antigenpositive donor); HCV antibody (positive donor is acceptable only if PCRfor viral DNA is negative); Herpes Simplex Virus antibody (documentstatus only; positive donor is not rejected); HIV I/II antibody (rejectHIV I/II positive donor); HIV PCR (reject HIV PCR positive donor); HIVI/II antibody (reject HTLV I/II positive donor); CMV antibody titer (ifpositive and recipient is negative, consider another donor if available,otherwise CMV screening and prophylaxis is mandatory); serologic testfor syphilis (if positive, perform a fluorescent treponemal antibodytest; donor is accepted if fluorescent treponemal antibody is negative);chest X-ray, if the donor is greater than 21 years of age; and EKG ifthe donor is greater than 40 years of age.

Example 5 Pre-Transplantation Treatment of Donor

For donors, a total of 560 cc of blood will be collected for archivingof lymphocytes for immunocompetence testing. This can be obtained as asingle blood donation pre-transplant (450 cc). The remaining eleven 10cc-yellow top collection tubes are obtained eight weeks after the firstdonation. For pediatric bone marrow donors, no more than 3 ml/kg at anyone time are drawn, and no more than 7 ml/kg over a six-week period aredrawn as per the NIH guidelines for pediatric research blood draws.

Beginning day −4 (with respect to HSC+hFC infusion) and for up to +4days, 10 ng/kg G-CSF is administered b.i.d. Collection begins on day −1.A minimum of 5×10⁶ CD34/kg total is collected. A maximum of twocollections are done. With each blood stem cell donation, 5-10 ml ofblood is taken at the start and at the end of the procedure to measureblood cell counts including enumeration of CD 34+ cells.

At two days and one week after donation, the donor is contacted toconfirm whether any adverse events have occurred. The donor also isasked to donate a blood sample (7 μl) one month after donation to ensureblood counts have recovered. The donor is treated with therapeutic iron,Vitamin K or calcium as needed. The visits for G-CSF administration,blood stem cell donations, and blood draws are summarized below in Table4 (e.g., an X marks what will occur on each visit).

TABLE 4 Blood Stem Symptom Filgrastim/ Cell Blood Visits AssessmentG-CSF Donation Draws Screening X X Preparation, Day −3 X X XPreparation, Day −2 X X Preparation, Day −1 X X Preparation, Day 0, X XX X First donation Second donation* X X X 2 days after donation X 1 weekafter donation X Potential blood draws X to test for donor chimerism inthe recipient (up to 3 years) *2nd donation occurs only if sufficientcells are not obtained in the 1st collection

Example 6 Recipient Conditioning

Individuals are examined by a radiation therapist to determine dosimetryfor TBI. Central venous access is established in all patients prior toinitiation of conditioning. Campath-1H is administered in a firstsession at day −53, −52, −51, and −50 at a maximum dose of 30 mg and ina second session at a maximum dose of 20 mg administered at day −24,−23, −22, and −21. The pediatric dose of Campath is 10 mg/day on cycleone and 7 mg/day on cycle two. For smaller recipients and those lessthan one year of age, Campath-1H is dosed at a rounded up dose of 0.4mg/kg for the first regimen, and at a rounded up dose of 0.3 mg/kg forthe second regimen. The route of administration of the Campath, eithersubcutaneously or intravenously, is at the discretion of the attendingphysician. Start dates for Campath administration can be moved forwardor backward 1-3 days to accommodate scheduling conflicts. Fludarabine isadministered on day −5, −4, and −3. The individual receives TBI andbegins cyclosporine immunosuppression at day −1. The secondimmunosuppressive medication, mycophenolate mofetil, is started theevening of HSC+hFC infusion (day 0). The conditioning regimen is shownin the following Table.

TABLE 5 Conditioning Approach Day −53 to −50 Campath-1H is administeredat 30 mg/day for adults and 10 mg/day for children over each of the fourdays. Day −24 to −21 Campath-1H is administered at 20 mg/day for adultsand 7 mg/day for children over each of the four days. Day −5 −4, −3Fludarabine is administered at 30 mg/m2 intravenously over a period of30 minutes on each of these three days. Day −1 Pre-transplantconditioning 200 cGy TBI (35-40 cGy/min); Cyclosporine is administeredday −1 and continued until it has been determined that the patient hasengrafted, or it has been demonstrated that the patient has failed toengraft, or at the discretion of the physician. If engraftment occurs,cyclosporine is continued for at least 12 months. If there is noengraftment, cyclosporine is discontinued. Marrow is processed to retainhFC and HSC using ferromagnetic approach. Day 0 HSC + hFC isadministered. MMF is started.

The radiation is delivered at day −1. The radiation dose is 200 cGy of 6MV accelerator X-rays, delivered in one fraction. A dose rate of 35-40cGy/minute is used, dependent on the distance, energy, and patientdimensions. Dose variations greater than 10% are evaluated and approvedon an individual basis. Infusion of the HSCs+hFCs occurs on day 0.Patients receive daily penicillin or equivalent prophylaxis for 2 yearspost-transplant, or longer at the discretion of the treating physician.

Example 7 HSC+hFC Cell Processing

The mobilized peripheral blood stem cells are incubated with monoclonalantibodies that are specific for alpha beta TCR T cells and B cells,then depleted by immunomagnetic separation. The composition of theinfused cells is assessed by immunofluorescent staining for CD34 HSCs;CD8+/TCR−/CD56^(dim/neg) hFCs; γδ T cells, and αβ-TCR+ T cells. Theadequacy of cellular depletion is determined by flow cytometricanalysis, and the clinician is notified of preliminary cell doses priorto infusion. The cell product also is analyzed for bacteria, fungus, andendotoxins. The HSC+hFC product is infused via a central venous line ina monitored setting per institutional guidelines.

The processed graft is administered to all subjects, and the graft isonly limited based on the maximal allowable alpha beta TCR dose.However, only those subjects with a minimally acceptable graft (e.g., atleast 5×10⁹ total leukocytes available from the collection to process;at least 5×10⁶ CD34/kg of recipient body weight; and a T cell depletionof less than 0.5 logs) are evaluated as described herein.

Example 8 Cell Dosing Algorithm

As many HSCs, hFCs and progenitors as possible are administered withinthe context of a maximal allowable T cell dose to avoid GVHD. Presently,the maximum dosing is 3.0×10⁶ to 4.2×10⁶ alpha beta T cells/kg recipientbody weight (with a preferred starting point at 3.8×10⁶ alpha beta Tcells/kg recipient body weight). Recipients are followed for a minimumof 28 days. If engraftment is not observed, the maximal allowable alphabeta TCR dose is increased by one unit (4×10⁵/kg recipient body weight).The maximal allowable alpha beta TCR dose is increased until stableengraftment is achieved without significant GVHD. For HLA-matchedtransplants, there is no maximum T cell cap and cell dose does notincrease based on the outcome of these matched transplants. For patientswho are mismatched, the maximal allowable alpha beta TCR dose isdetermined

TABLE 6 HSC, hFC, progenitors As many as possible NK cells, B cellsRecord and report doses γδ-TCR+ T cells Record and report doses αβ-TCR+T cells For HLA matched, there will be no cap. For HLA mismatched, themaximal allowable will be determined by the last safe dose in thekidney, heart, liver tolerance, sickle cell, and MS protocols.

If significant (>0.5%) donor engraftment is observed in the first 28days, the individual is followed for an additional 28 days to assess theincidence of acute GVHD.

Example 9 Additional Sickle Cell Patients Transplanted

Subject #5 was 9 years of age at the time of transplant (March 2006). Hehad experienced multiple pain crises, two episodes of acute chestsyndrome before transplant, and had been treated with exchangetransfusions for 7 years. The subject received HLA-matched trait siblingdonor's iliac crest bone marrow and was conditioned with essentially thesame regimen as described in Section C above. The graft contained5.24×10⁶ CD34+ cells/kg of body weight, 0.55×10⁶ alpha beta-TCR⁺cells/kg body weight and 0.35×10⁶ FC cells/kg body weight. The subjecthas been transfusion-independent post-stem cell transplant with 100%donor RBC production and chimerism levels at 20-30% donor by FISH forgreater than 1525 days post-transplant. Immunosuppression wasdiscontinued at 23 months post-transplant. Subject has not exhibitedgraft-versus-host disease (GVHD), transplant-related toxicity, or sicklecell complications since transplant.

Subject #7 was a 16-year-old male who experienced repeated acute chestsyndrome episodes that required red blood cell transfusion therapy.Prior to undergoing the transplant in September 2009, he washospitalized for osteomyelitis of the right knee and multiplevaso-occlusive painful events. The subject received a haploidenticaltransplant from his parent. The subject was conditioned essentially asdescribed above in Section C. The subject tolerated the conditioningwell and the transplant was uneventful. He received 3.26×10⁶ CD34+cells/kg body weight, 3.8×10⁶ alpha beta TCR+ cells/kg body weight, and0.5×10⁶ FC cells/kg body weight, and he was managed as an outpatient.Unfortunately, this subject was not compliant in the immediatepost-transplant period and did not regularly take cyclosporine and MMFas required. Chimerism was not present at post-transplant months 1 and2. Post-transplant, he experienced a recurrent pain crisis thatsubsequently resolved. The subject remains on the study to monitor foradverse events, but chimerism testing was discontinued afterpost-transplant month two.

Subject #8 was a 12-year-old female who experienced numeroushospitalizations for pain crises. She also had undergone a splenectomyfollowing sequestration and cholcystectomy. She underwent conditioningessentially as described above in Section C, and she received ahaploidentical transplant from her parent, who had the SCD trait. Shereceived 19.1×10⁶ CD34+ cells/kg body weight, 3.8×10⁶ alpha beta TCR+cells/kg body weight, and 0.79×10⁶ FCs/kg body weight, and, followingtransplantation, she was managed as an outpatient. She tolerated theconditioning very well and demonstrated robust donor engraftment of 71%at one month post-transplant. Her whole blood chimerism remained durableat 84%, with lymphoid chimerism at 58% and myeloid chimerism at 95% atmonth nine. She was producing 100% donor RBC as reflected by hemoglobinA at 57%, hemoglobin S at 41%, and hemoglobin A2 at 2% as demonstratedby hemoglobin electrophoresis. The subject has not required transfusiontherapy since transplant and is asymptomatic. She has had no evidencefor GVHD.

Subject #9 was a 25-year-old male who experienced repeated PRBCtransfusions, cholecystectomy with sickle cell disease, hypertension andrenal vascular disease prior to transplant. The subject was conditionedessentially as described above in Section C, and he tolerated theconditioning well. The alpha beta TCR+ cells for this subject wasincreased to 4.2×10⁶ cells/kg body weight, and the subject also received1.46×10⁶ CD34+ cells/kg body weight and 0.72×10⁶ FCs/kg body weight. Hedemonstrated 10% donor chimerism at post-transplant month one. Hischimerism decreased to 4% at month two and to less than 2% at day 100.The subject was admitted for elevated creatinine due to calcineurininhibitor (CNI) sensitivity in the second post-transplant month. Thedose was adjusted and the SAE resolved. About one month later, he wasadmitted for fever, gram positive cocci, and CMV infection. He went offstudy to participate in an investigational drug for CMV treatment.

Section E Prevention of Graft vs. Host Disease (GVHD) Following SolidOrgan Transplant Example 1 Patient Recruitment

Candidates for the protocol were selected from the list of patientsawaiting renal transplantation or who were being evaluated fortransplantation. This selection process was carried out by thetransplant surgeons and the transplant nurse coordinators of theInstitute of Cellular Therapeutics at the University of Louisville (“theInstitute”).

Example 2 Inclusion Criteria

A candidate patient must be between the ages of 18 and 65 years and meetthe Institution's criteria for renal transplantation for end-organfailure. A candidate patient must be receiving his or her first renaltransplant. A candidate patient must be receiving a renal transplantonly. The crossmatch must be negative between the donor and therecipient. Women who are of child bearing potential must have a negativepregnancy test (urine test is acceptable) within 48 hours prior toinitiating TBI and must agree to use reliable contraception for 1 yearfollowing transplant. Candidate patients must exhibit no evidence ofdonor-specific antibody, presently or historically.

Example 3 Exclusion Criteria

Patients are not candidates if they have a clinically active bacterial,fungal, viral or parasitic infection, or if they are pregnant. Patientsare not eligible if they exhibit clinical or serologic evidence of viralinfection that would preclude the recipient from receiving a kidneytransplant. A patient is not a candidate if they have received previousradiation therapy at a dose which would preclude TBI, if there is apositive crossmatch between the donor and the recipient, or if there isevidence for immunologic memory against the donor. Patients also areexcluded if their body mass index (BMI) is less than 18 or greater than35.

Example 4 Donor Selection Criteria

Donors for this protocol must meet all of the Institute's criteria forrenal and stem cell transplant.

Example 5 Protocol

The timing for all manipulations is relative to the TBI conditioning ofthe recipient on day 0. Beginning day −3 and for up to four days, 10ng/kg G-CSF was administered b.i.d. Collection began on day 0. On day 0,a CD34 count was performed prior to giving the final dose of G-CSF.HSC+hFC transplantation was scheduled 4 to 6 weeks prior to the desireddate of kidney harvest. The donation and transplant of the kidney is notschedules until the donor's platelet counts have returned to baselineand safe levels for kidney donation (e.g., greater than 100,000/n1 ofwhole blood).

The visits for G-CSF administration, blood stem cell donations, andblood draws are summarized in Table 7 The ‘X’ marks what will occur oneach visit.

TABLE 7 Donor Mobilization Blood Stem Symptom Filgrastim/ Cell BloodVisits Assessment G-CSF Donation Draws Screening X X Preparation, Day −3X X X Preparation, Day −2 X X Preparation, Day −1 X X Preparation, Day0, X X X X First donation 2 Days after donation X 1 Week after donationX Potential blood draws X to test for donor chimerism in the recipient(up to three years)

Example 6 Pre-Transplant Conditioning

Cell dose (HSC+hFC) as well as degree and type of conditioning of therecipient were independent variables that influence engraftment. In thecurrent protocol, cell dose and conditioning were optimized until >1%donor chimerism was established. The initial target cell dose forHSC+hFC was ≧1×10⁸ CD34+/kg. The first patients received 200 cGy TBI,fludarabine (30 mg/m² day −3 to −1), and post-transplantimmunosuppression with MMF (15 mg/kg q 12 h beginning day 0), and FK506(0.02 mg/kg q 12 h beginning day −1) for six months, or as clinical needrequired. The decision to use either FK506 (tacrolimus) or cyclosporinewas left to the physician because patients differ in their ability totolerate either drug. The marrow was infused on day +1. The schedule isshown in Table 8.

TABLE 8 Day Treatment Dose −3 Fludarabine after dialysis (if required)30 mg/m2 −2 Fludarabine after dialysis (if required) 30 mg/m2 −1Fludarabine 30 mg/m2 0 Start MMF and FK506 or cyclosporine 0 TBI (200cGy) 35-40 cGy/min Harvest donor marrow and process to obtain HSC + hFC+1 Infuse HSC + hFC after dialysis (if required) +28-60 Renaltransplantation; continue MMF and calcineurin inhibitors

If the recipient required dialysis, the dosing of fludarabine and theHSC+hFC infusion occurred after dialysis on the specified days. On themorning of HSC+hFC infusion, an extra liter of volume was removed fromdialysis to account for the volume of HSC+hFC. Dialysis then wasscheduled for 48 hrs or later following HSC+hFC infusion to give thecells an optimum opportunity to home to the marrow compartment.

Example 7 Outcomes

A minimum of about 2 weeks post-HSC+hFC infusion or as long as therecipient needs to fully recover from the stem cell transplant procedurepassed before the renal transplant was performed from the same donor.The following algorithm was used based on the outcome of the HSC+hFCtransplant.

1) if the recipient exhibited chimerism of ≧1% and was determined to betolerant to the donor, at least six months of Prograf and MMF wasadministered while donor:host tolerance to the kidney is established.The recipient did not receive Campath-1H or any additionalimmunosuppression at the time of transplant.

2) if the recipient did not engraft, the patient as tested by flowcrossmatch prior to transplant to ensure no donor-specific antibodiesdeveloped. If no donor-specific antibody was present, the patientunderwent living donor kidney transplant using conventionallymphodepletion induction with Campath-1H followed by maintenanceimmunosuppression with FK506 and MMF. Other lymphodepletion approachesfor induction therapy such as ALG can be used in place of Campath perstandard of care.

3) if donor-specific antibody developed, the patient was assessed and aclinically appropriate antibody reduction protocol implemented prior totransplantation. Patient sensitization was not expected.

Example 8 Cell Dosing Algorithm

The goal of the study was to engineer a graft with adequate HSCs, hFCsand progenitors for allogeneic engraftment while avoiding GVHD. A celldosing algorithm was established that is tied to the maximum allowablealpha beta T cell dose. For example, if toxicity (GVHD) did not occurbut engraftment was not durable, the maximum allowable T cell dose wasincreased by 1 unit (see below). The maximal allowable T cell dosecontaining as many HSCs, hFCs, and progenitors was administered. Thealgorithm that was used is shown in Table 9.

TABLE 9 HSC, hFC, As many as possible progenitors alpha beta For HLAmatched, there is no cap. For TCR+ T cells HLA mismatched, the maximalallowable is determined by the last safe dose in the kidney, heart,liver tolerance, sickle cell, and MS protocol NK cells, B Record andreport doses cells gamma delta Record and report doses TCR+ T cells

The present cell dosing currently allows a maximum of 3.0×10⁶ to 4.2×10⁶alpha beta T cells/kg recipient body weight; 3.8×10⁶ alpha beta Tcells/kg recipient body weight was the starting dose. Each patient wasfollowed for at least 28 days. If no evidence of engraftment wasobserved, the maximal allowable alpha beta-TCR dose was increased by oneunit (4×10⁵/kg recipient body weight). The maximal allowable alphabeta-TCR dose was increased in subjects until stable engraftment wasachieved without significant GVHD. For HLA-matched transplants, therewas no maximum T cell cap and cell dose was not increased based on theoutcome of the matched transplants. For patients who are mismatched, themaximal allowable alpha beta-TCR dose was determined.

If significant (>0.5%) donor engraftment was observed in the first 28days, the subject was followed for an additional 28 days to assess theincidence of acute GVHD before the cell dose was increased. It wasexpected that the majority of cases of acute GVHD would be apparent by 8weeks post-transplant. If evidence of severe GVHD was seen, the maximalallowable T cell dose was reduced to a level proven safe. To date, GVHDhas not been observed.

Example 9 HSCs+hFCs

Donor PBMC was processed to enrich for hFCs and HSC. Approximately 85%of total bone marrow composition was removed, including GVHD-producingT-cells and B-cells, using a ferromagnetic approach. The resultantproduct was enriched for hFCs, HSCs and progenitors. After the adequacyof the processing was confirmed by flow cytometry, the HSC+hFC graft wasapproved for infusion. A delay in the bone marrow transplantation of upto 72 hours following donor cell harvest was accepted to allow for bonemarrow processing and transplantation. Dose-adjusted Bactrim and Valcyte(if CMV+) or Valtrex (if CMV−) prophylaxis was started. The patient wascarefully monitored during and after infusion of marrow to detect anychanges in, for example, respiration, blood pressure, or angioedema,which may be an indication of hypersensitivity.

Example 10 Post-Transplant Immunosuppression

Subjects enrolled in this protocol received standard immunosuppressionat the discretion of the attending physician and according toinstitutional protocol. For deceased donor kidney/HSC+hFC recipients andliving donor HSC+hFC recipients without demonstratable donor chimerismat 1 month, this generally included Prograf plus MMF afterlymphodepletion induction therapy with ALG or Campath. Prograf levelswere maintained between 8-12 ng/ml. Starting on day 0, MMF was generallydosed at 1-1.5 gms b.i.d. A one time dose of Campath, 30 mg IV, wasoptionally given in the operating room. SoluMedrol was given at a doseof 500 mg IV in the operating room one hour prior to the Campath dose,then post-operatively on day 1 at a dose of 250 mg IV and on post op day2 at a dose of 125 mg. FK506 and MMF were continued for at least 6months in patients who were chimeric to promote engraftment andtolerance induction.

Example 11 Preliminary Solid Organ Transplant Protocol

Preliminary experiments demonstrated the success and safety of HSCs+hFCsand immune-based nonmyeloablative conditioning in renal transplantrecipients. Dosing of the HSCs+hFCs was performed to maximize safety andoptimize HSC and hFC content. The overall goal was to completely avoidGVHD. Solid organ tolerance protocols were begun with a maximum of0.2×10⁶ T cells/kg recipient body weight to perform a dose-escalation.Total alpha beta T cells were used in the dose-escalation experimentssince the other effector cells for GVHD (NK, B-cells, and APC) are allpresent in amounts proportional to total alpha beta T cells. CD34 andhFC dose were optimized within the maximum allowable T cell dose. Nosignificant immunologic events (i.e., rejection episodes or antibodyproduction) were observed in any of the patients. None of the patientsdeveloped GVHD, but only transient chimerism was observed.

Since September 2004, nine heart and eleven kidney patients have beentransplanted using the dose-escalation strategy set forth in Table 10.Patients 5, 6, 12, and 13, which are highlighted in Table 10, aredescribed in more detail; three patients underwent simultaneouskidney/HSC+hFC transplantation from living donors and the remainingpatient received his kidney from a deceased donor. All four patientswere conditioned with 200 cGy TBI and underwent lymphodepletioninduction therapy with Campath followed by maintenance immunosuppressionwith MMF and a calcineurin inhibitor. They did not receive fludarabine.The 4 patients are briefly described below.

Patient #5 is a 55-year-old male who underwent a deceased donor renalallograft/HSC+hFC transplant in September 2005. The patient received3.7×10⁶ CD34 and 0.8×10⁶ hFC per kg recipient body weight. Theconditioning was well tolerated and no adverse events related to theapproach occurred. The donor and recipient shared a 1/6 HLA-antigenmatch. The patient experienced the anticipated nadir, and then recoveredimmune function and endogenous hematopoiesis. He is doing well with arecent serum creatinine of 2.1.

Patient #6 is a 58-year-old male who underwent a living donorkidney/HSC+hFC transplant from an unrelated friend in November 2005. Thepatient received 1.33×10⁶ CD34 and 0.18×10⁶ hFC per kg recipient bodyweight. The conditioning was well tolerated and no adverse eventsrelated to the approach occurred. The donor and recipient shared a 1/6HLA-antigen match. The patient experienced the anticipated nadir, andthen recovered immune function and endogenous hematopoiesis. He is doingwell, with a recent serum creatinine of 2.0.

Patient #12 is a 37-year-old female who underwent a living donorkidney/HSC+hFC transplant from her cousin in October 2007. She received2.24×10⁶ CD34 and 0.41×10⁶ hFC per kg recipient body weight. Theconditioning was well tolerated and no adverse events related to theapproach occurred. The donor and recipient shared a 2/6 HLA-antigenmatch. The patient experienced the anticipated nadir, and then recoveredimmune function and endogenous hematopoiesis. She is doing well with arecent creatinine of 1.2.

Patient #13 is a 49-year-old female who underwent a kidney/HSC+hFCtransplant from her brother in November 2007. The patient received3.85×10⁶ CD34 and 0.78 10⁶ hFC per kg recipient body weight. Theconditioning was well tolerated and no adverse events related to theapproach occurred. The donor and recipient shared a 4/6 HLA-antigenmatch. The patient experienced the anticipated nadir, and then recoveredimmune function and endogenous hematopoiesis. She is doing well with arecent creatinine of 1.2.

TABLE 10 Cell dosing for patients (per kg recipient weight)

*× 10⁶ cells; VB = vertebral body; MPB = mobilized peripheral blood; IC= iliac crest

Example 12 Results of Preliminary Protocol

Initially, the chimerism observed was low (<0.2%) and only transient.However, as the total cell dose was increased, durable mixed chimerismwas achieved. The immune response to the graft was modulated by themarrow infusion as evidenced by transient donor-specific tolerance inmixed lymphocyte reaction (MLR) assays observed in the more recentlytransplanted patients and the absence of any clinical or histologicrejection episodes.

The fact that endogenous hematopoiesis resumed in those patients who didnot engraft confirmed the non-myeloablative nature of the conditioning.There was an expected nadir of absolute neutrophil count (ANC) of lessthan 1,000 that occurred in the recipients between 7 and 18 days (FIGS.9A and 11A), which was managed as an outpatient in Patient #12. It wasfound that administration of G-CSF did not accelerate recovery. The MMFand FK506 was continued through the nadir to promote engraftment.Recovery of B cells (CD19), CD4+ cells, and CD8+ cells occurred by 3months in the Campath-lymphodepleted recipient #12 (FIG. 11B). Plateletcounts were determined following solid organ transplant (FIGS. 9B, 10A,and 11C). The platelet nadir, if present, typically was brief andusually did not require transfusion therapy. The chimerism that wasestablished in solid transplant patients is shown in FIGS. 9C and 10B.

Example 13 Modified Protocol for Solid Organ Transplant

The kidney/HSC+hFC transplant protocol was modified to add fludarabineconditioning and to perform the living donor transplants sequentially,with HSC+hFC administered one month prior to the kidney graft placement.

The first transplant (stage 1 FCRx) was performed in March 2008 (Patient#15 in Table 10). She is a 31-year-old female whose husband was herdonor. The patient is currently in her nadir period and is doing well asan outpatient. A flow crossmatch performed on day 14 was negative (Table11). In this assay, the binding of antibodies to donor T and B cells wasmeasured by flow cytometric analysis in MCDF units.

TABLE 11 Flow Crossmatch T cell B cell % T Positive/ % B Positive/ cellsMCDF Negative cells MCDF Negative Negative 57 250 − 5 301 − controlPositive 52 495 + 5 534 + control Patient #15 54 245 − 5 246 − Patient#15 52 252 − 5 266 −

These results demonstrated the safety of the non-myeloablativeconditioning and the feasibility of the HSC+hFC process and product insolid organ transplant. It is noted that none of the recipients becamesensitized to the donor.

Section F Kidney Transplant Example 1 Donor and Recipient Eligibility

All protocols were approved by the Northwestern Institutional ReviewBoard, the FDA IND 13881, and informed consent was obtained for alldonors and recipients. Donors and recipients had to meet Institutionalcriteria as suitable living transplant donors and recipients;participants had to complete all phases of the pre-transplant donor andrecipient evaluation to be considered for study participation. Inclusioncriteria for transplant recipients included age between 18 and 65 years,absence of any donor-specific antibodies as assessed by flow PRAanalysis, and receiving only a living donor kidney transplant. Women ofchildbearing age had to have a negative pregnancy test (urine testingacceptable) within 48 hours of receiving TBI and agree to use reliablecontraception for a year after the transplantation. Exclusion criteriaincluded clinically active bacterial, fungal, viral or parasiticinfection, pregnancy, previous radiation therapy at a dose which wouldpreclude TBI, a positive flow cytometry crossmatch between donor andrecipient, presence of donor-specific antibodies, body mass index(BMI)>35 or <18, and positive serologies for HBV, HCV, and HIV.

Example 2 Conditioning and Donor Product Preparation

Conditioning consisted of three doses of fludarabine (30/mg/kg/dose) atdays −4, −3, −2; two doses of cyclophosphamide (Cytoxan; 50 mg/kg/dose)at days −3 and +3; and 200 cGy TBI at day −1 relative to the renaltransplant as depicted in FIG. 12. Hemodialysis was performed 6-8 hafter the administration of fludarabine and Cytoxan. Tacrolimus (targettrough concentrations 8-12 ng/ml) and mycophenolate mofetil (MMF)(Cellcept; 1 gm orally twice daily if recipient weighs <80 kg, 1.25 gmtwice daily if recipient weighs >80 kg) were started at day −3 andcontinued throughout. HSCs+hFCs can be administered to the recipient atday 0 (i.e., the same day as the transplant) or at day +1.

Example 3 Hematopoietic Stem Cell Collection

At least two weeks prior to the renal transplant, donors were mobilizedwith granulocyte colony stimulating factor (G-CSF) at 10 mcg/kg b.i.d.and apheresis was performed on day +4. The product was transported bycourier to the Institute for Cellular Therapeutics (ICT) and processedto remove mature graft-versus-host disease (GVHD)-producing cells whileretaining hematopoietic stem cells (HSC), facilitating cells (FCs), andprogenitor cells. The product was then shipped back to NorthwesternUniversity for infusion, either as a fresh product or cryopreserved.

Example 4 Immunologic Monitoring

The recipient response to PHA, Candida, tetanus toxoid, donor andthird-party alloantigens was tested monthly (see, for example, Patel etal., 2008, J. Allergy Clin. Immunol., 122:1185-93). Flow crossmatchassay to detect donor antibodies were performed at 1 and 6 months.Chimerism testing was performed by molecular assay using short tandemrepeats (Akpinar et al., 2005, Transplant., 79:236-9). Surveillancebiopsies were performed at 1 year. At selected time points,immunophenotypic analysis of peripheral blood was performed for T cell,B cell, NK cell, monocytes, CD4⁺/CD25⁺ Fox P3⁺ regulatory T cell(T_(reg)), and T effector cell (T_(eff)) recovery.

Example 5 Chimerism Testing

Chimerism was determined by genotyping of simple sequence-lengthpolymorphisms encoding short tandem repeats (STR). For lineage chimerismtesting, CD 19⁺ (B cells), CD3⁺ (T cells), and CD66B⁺ myeloid cells weresorted from whole blood then analyzed by molecular STR typing.

Example 6 Weaning of Immunosuppression

Prograf and MMF were continued per standard of care until 6 monthspost-transplant. At that point, if chimerism or donor-specific tolerancewere present, the MMF was first discontinued, then the Prograf wastapered off over to sub-therapeutic amounts the next few months (e.g.,<3.0 ng/ml by 9 months). Prograf was discontinued at 12 months ifevidence of chimerism and/or in vitro donor specific hyporesponsivenessis present.

Example 7 Results

A summary of Subject #1-Subject #9 is shown below in Table 12, and Table13 shows the cell dosing regimens. A few of the subjects are discussedin more detail as follows.

Subject #3 is a 43-year-old white male who developed ESRD due topolycystic kidney disease. A 1-of-6 HLA matched unrelated altruist washis donor. A total of 3.8×10⁶ alpha beta TCR⁺ T cells, 2.53×10⁶ CD34cells, and 4.48×10⁶ FC/kg recipient body weight cryopreserved productwere infused. The recipient demonstrated 95% donor chimerism at 1 month,and chimerism fluctuated between 63% and 100% over 18-monthspost-transplant (FIG. 13A). At 12 months, multilineage testing revealed100% B cell, T cell, and myeloid production (FIG. 13B). Flow crossmatchwas negative at 1 month and 6 months. At month 5, the recipientexhibited donor-specific tolerance and immunocompetence to respond tothird-party alloantigen (FIG. 13C). This has persisted through 12months. His renal function has remained stable based on creatinineoutput (FIG. 13D). The subject exhibited a transient nadir between 6-15days (FIGS. 13E and 13F), which was managed as an outpatient.

Subject #5 is a 40-year-old male whose renal failure was secondary tochronic glomerulonephritis. He underwent a combined FC/renal transplantfrom a 1-of-6 HLA matched unrelated donor. His product was comprised of3.8×10⁶ alpha beta-TCR⁺ T cells, 0.7×10⁶ FC, and 3.94×10⁶ CD34 cells/kgrecipient body weight. The nadir followed a pattern similar to the priorsubject. Chimerism was 100% at 1 month, 92% at 3 months, and 94% atmonth 5. A donor-specific tolerant profile began to emerge at month 3,with responses to PHA and third-party alloantigen but not to donor.

Subject #6 is a 39-year-old female who developed ESRD secondary toreflux. She underwent a second renal transplant from a 2-of-6 HLAmatched unrelated donor. The product consisted of 3.8×10⁶ alpha betaTCR⁺ T cells, 8.59×10⁶ CD34⁺, and 3.11×10⁶ FC cells/kg recipient bodyweight. The recipient exhibited 100% donor chimerism at 1 month.

TABLE 12 Summary of Kidney + hFC Patients HLA Date of Subject Sex AgeMatch Transplant Original Disease Adverse Events 1 M 50 5/6 February2009 Membranous recurrent disease at 1-yr post-transplant; successfullytreated with rituximab 2 M 56 3/6 April 2009 Hypertension febrile septicepisode at 3-months post- transplant, marrow failure, autologous HSCTrescue, sepsis and allograft failure; now successfully retransplantedwith living donor kidney 3 M 43 1/6 May 2009 PCKD drug rash, shingles 4M 29 3/6 June 2009 Alports wound infection, subclinical rejection atSyndrome one-year post-transplant 5 M 40 1/6 February 2010 Chronic GNflank cellulitis, wound seroma 6 F 39 2/6 March 2010 Reflux: 2^(nd) noneTransplant 7 M 35 3/6 April 2010 Hypertension none 8 F 46 1/6 July 2010PCKD i.v. site cellulitis 9 M 28  0/6* September 2010 IgA Nephropathyhemolytic uremic syndrome due to FK506, converted to sirolimus andresolved *1 minor antigen match

TABLE 13 Cell Dosing for Patients Composition delivered (10⁶/kgrecipient weight) Anti-donor alpha % chimerism Antibody beta T PatientSource* at 1 month Production cells CD34 FC 1 IC* 30 No 0.963 .896 0.1572  MPB* 95 No 3.8 2.53 4.48 3 MPB 100 No 3.8 3.6 0.90 4 MPB 25 No 1.941.00 0.49 5 MPB 100 No 3.8 3.94 0.716 6 MPB 100 No 3.8 8.59 3.11 7 MPB100 No 3.8 16.9 1.16 8 MPB 100 NA 3.8 12.6 2.74 9 MPB 0 NA 3.8 5.07 2.12*Source: IC, iliac crest marrow; MPB, mobilized peripheral blood

Example 8 Summary of hFC and Living Donor Kidney Transplant

Of the 9 subjects transplanted, the non-myeloablative conditioning waswell-tolerated. In addition, the post-transplant nadir period for allsubjects was easily managed as an outpatient.

Eight of the nine subjects demonstrated macrochimerism followingtransplantation, ranging from 6% to 100% at 1-month. Durable chimerismwas achieved in the majority of subjects.

One subject has been weaned entirely off of immunosuppression. Severalsubjects have exhibited evidence of donor-specific hyporesponsivenessand are poised to be weaned from immunosuppression. Subjects wereimmunocompetent to respond to mitogen (PHA), Candida, and MHC-disparatethird party alloantigen. None of the subjects developed GVHD despite theHLA mistatching.

Section G Metabolic Disorders Example 1 Treatment of Inherited MetabolicDisorders

Subject #1 was a seven-year-old child with metachromatic leukodystrophy.He received a 3 out of 6 HLA-matched transplant from his father, whocarries the trait for metachromatic leukodystrophy. The subject wasconditioned essentially as described above in Section C, Example 2. Hetolerated the conditioning and infusion very well as an outpatient. Hereceived 14.4×10⁶ CD34+ cells/kg body weight, 3.8×10⁶ alpha beta TCR+cells/kg body weight, and 4.1×10⁶ FCs/kg body weight. His nadir wasbrief and he did not require transfusion therapy. His chimerism, bymolecular STR, has ranged between 80%-98%. At 14 months post-transplant,the recipient exhibited no GVHD. The MLR results for this subjectdemonstrated tolerance to the donor and confirmed the likelihood ofdurable long term engraftment. Pre-transplant, the subject'sArylsulfatase A enzyme level was 3, compared to the donor's level of 50post-transplantation. The subject's level was approximately 50 at threeand six months post-transplant, and was 88.6 at one yearpost-transplant. This represents the enzyme level of a phenotypicallynormal patient.

Other Embodiments

It is to be understood that while the methods and compositions has beendescribed in conjunction with the detailed description thereof, theforegoing description is intended to illustrate and not limit the scopeof the invention, which is defined by the scope of the appended claims.Other aspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A cellular composition comprising at least about30% human facilitating cells (hFCs), wherein said hFCs comprise cellshaving a phenotype of CD8+/alpha beta TCR−/CD56^(dim/neg) and cellshaving a phenotype of CD8+/alpha beta TCR−/CD56^(bright).